Conservation of Salmonella typhimurium virulence plasmid maintenance regions among Salmonella serovars as a basis for plasmid curing.

The association of large plasmids with virulence in invasive Salmonella serovars has led to a number of studies designed to uncover the role of these plasmids in virulence. This study addresses two aspects of virulence-associated plasmids. The first is the distribution of the replication and maintenance regions among the plasmids of different Salmonella serovars, and the second is the use of the conserved virulence plasmid par region to provide a rapid method for eliminating the virulence plasmids specifically. Colony blots revealed that the par and repB regions of the S. typhimurium virulence plasmid hybridized with 80% of the isolates of S. choleraesuis, S. dublin, S. enteritidis, S. gallinarum, S. pullorum, and S. typhimurium, while the repC region was not detected in any of the isolates of S. dublin, S. gallinarum, or S. pullorum. None of these maintenance regions was found in any of the 30 additional serovars tested. The large plasmids of those serovars that hybridized with par were labeled with a Kmr insert within parA via P22HTint or P1L4 transduction, which destabilized the plasmids and allowed the rapid isolation of plasmid-free derivatives for all of the serovars, except for S. dublin, which exhibited weak homology with par.     

Molecular epidemiology of a nosocomial outbreak due to SHV-4-producing strains of Citrobacter diversus.

Over a 6-month period, eight strains of Citrobacter diversus (Citrobacter koseri) resistant to extended-spectrum cephalosporins and monobactams were isolated from seven colonized and/or infected patients from the same intensive care unit. All strains harbored a single large conjugative plasmid which mediated an extended-spectrum beta-lactamase of the SHV-4 type (ceftazidimase phenotype; enzyme pI, 7.8; plasmid DNA hybridization with a blaSHV-specific probe). All strains were characterized by antibiotic resistance pattern analysis, beta-lactamase content analysis, plasmid profiling, ribotyping with EcoRI, and arbitrarily primed (AP)-PCR with primers O8 and O12. Among the eight C. diversus strains, strains Cd5 to Cd12, six isolates (isolates Cd6 to Cd11) were identical by all markers; one strain (strain Cd5) differed by two markers (antibiotype and AP-PCR pattern with primer O8), and the remaining strain (strain Cd12) differed by two other markers (ribotype and AP-PCR pattern with primer O12). Our results suggest that six of the eight SHV-4-producing C. diversus strains studied (strains Cd6 to Cd11) were a single epidemic strain. Strain Cd5 could be related to the epidemic strain; the origin of strain Cd12 remains uncertain.     

Value of plasmid profiling, ribotyping, and detection of IS200 for tracing avian isolates of Salmonella typhimurium and S. enteritidis.

Seventy selected strains of Salmonella typhimurium and S. enteritidis isolated from related poultry flocks in three independent geographical areas were characterized by phenotypic and genotypic methods to compare the usefulness of the methods in epidemiological studies. The 56 S. typhimurium isolates were poorly discriminated by their biotypes, resistance patterns, and plasmid profiles. Nine different ribotypes were obtained after DNA digestion by BglII, PvuII, and SmaI. Seven IS200 types, characterized by six to nine copies of IS200 on the chromosome, were detected after digestion of genomic DNA by PstI. These studies resulted in the definition of 15 clonal lineages distributed in three clusters. The 14 S. enteritidis strains were not discriminated either by ribotyping or by detection of IS200 (IS200 typing), but were separated on the basis of antibiotic resistance and plasmid profiling. The stability of the insertion sequence type was confirmed by inoculation of an S. typhimurium strain to axenic chickens reared for 15 weeks in sterile isolators.     

Characterization of murine antibody response to Salmonella typhimurium by a class-specific solid-phase radioimmunoassay.

A heavy-chain class-specific, solid-phase radioimmunoassay was developed to characterize the murine antibody response to Salmonella typhimurium. The specificity of the assay was verified by quantitation of the extent of binding of anti-S. typhimurium antibodies to other bacterial genera and species and by cross-adsorption studies. The sensitivity of the procedure was also examined, and it was determined to be substantially more sensitive than either the passive hemagglutination or the whole-cell agglutination technique. The method was subsequently used to analyze th murine antibody response to S. typhimurium. Groups of mice were prebled and then immunized with live S. typhimurium via different routes. The animals were bled weekly for 12 weeks, and then sera were assayed for antibodies directed against whole bacteria or purified lipopolysaccharide. Anti-Salmonella antibodies of the immunoglobulin M class appeared in the serum approximately 2 to 3 weeks after immunization, and then immunoglobulin G anti-Salmonella antibodies appeared which constituted the major part of the long-term response. Immunoglobulin A was not a major component of the serum antibody response. The antibodies were primarily directed against the lipopolysaccharide determinants, but a small percentage of the response was directed against other cell surface components. Qualitatively and quantitatively similar anti-Salmonella antibody responses were observed in sera of outbred and inbred strains of mice.     

Characterization of intestinal invasion by Salmonella typhimurium and Salmonella dublin and effect of a mutation in the invH gene.

The relative levels of invasiveness of two bovine isolates each of Salmonella typhimurium and Salmonella dublin and of invH mutants of S. typhimurium were determined in MDCK and Int 407 cultured-cell assays and in bovine ileal loops. S. dublin was found to be significantly less invasive in cultured cells than S. typhimurium, but this difference was not observed in bovine intestines. The invH mutants exhibited a significant reduction in invasion in both cultured cells and bovine intestines. The invasive phenotypes of the strains were confirmed by fluorescent microscopy and scanning and transmission electron microscopy. The wild-type strains were observed in the laminae propriae of the intestinal villi, while in contrast the invH mutants were generally associated with the enterocyte layer. The degree of damage in the bovine ileum was related to the magnitude of the invasion. There was no difference in the amount of S. typhimurium or S. dublin recovered from the bovine ileum either with or without Peyer's patches 3 h after inoculation of the loop.     

Comparison of plasmid profile analysis, phage typing, and antimicrobial susceptibility testing in characterizing Salmonella typhimurium isolates from outbreaks.

We compared the phage types, antimicrobial resistance patterns, and plasmid profiles of 20 groups of isolates received at the Centers for Disease Control from Salmonella typhimurium outbreaks between 1975 and 1982 to determine the most useful laboratory method for identifying epidemiologically related isolates of S. typhimurium. In 18 (90%) of the 20 outbreaks, epidemiologically related isolates were identified as being the same by each of the three methods. In a subgroup of nine outbreaks in which isolates unrelated to the outbreak were submitted for comparison, outbreak isolates were differentiated from such control isolates six times (67%) by phage typing alone, four times (44%) by antimicrobial susceptibility testing alone, and eight times (89%) by plasmid profile analysis alone. Epidemic isolates were multiply susceptible, nontypable, or without plasmids in 14 (70%), 1 (5%), and 3 (15%), respectively, of the 20 outbreaks. Plasmid analysis appeared to be at least as specific as phage typing in identifying epidemiologically related isolates of S. typhimurium as being the same or in differentiating them from control specimens; both techniques appeared to be superior to antimicrobial susceptibility testing.     

Analysis of molecular epidemiology of Chilean Salmonella enterica serotype enteritidis isolates by pulsed-field gel electrophoresis and bacteriophage typing

Human Salmonella enterica serotype Enteritidis infections emerged in Chile in 1994. S. enterica serotype Enteritidis phage type 1 isolates predominated in the north, and phage type 4 isolates predominated in the central and southern regions. A study was planned to characterize this epidemic using the best discriminatory typing technique. Research involved 441 S. enterica serotype Enteritidis isolates, including clinical preepidemic samples (n = 74; 1975 to 1993) and epidemic (n = 199), food (n = 72), poultry (n = 57), and some Latin American (n = 39) isolates. The best method was selected based on a sample of preepidemic isolates, analyzing the discriminatory power (DP) obtained by phage typing and randomly amplified polymorphic DNA and pulsed-field gel electophoresis (PFGE) analysis. The highest DP was associated with BlnI PFGE-bacteriophage typing analysis (0.993). A total of 38 BlnI patterns (B patterns) were identified before the epidemic period, 19 since 1994, and only 4 in both periods. Two major clusters were identified by phylogenetic analysis, and the predominant B patterns clustered in the same branch. Combined analysis revealed that specific B pattern-phage type combinations (subtypes) disappeared before 1994, that different genotypes associated with S. enterica serotype Enteritidis phage type 4 had been observed since 1988, and that strain diversity increased before the expansion of S. enterica serotype Enteritidis in 1994. Predominant subtype B3-phage type 4 was associated with the central and southern regions, and subtype B38-phage type 1 was associated with the north (P < 0.0001). Food and poultry isolates matched the predominant S. enterica serotype Enteritidis subtypes, but isolates identified in neighboring countries (Peru and Bolivia) did not match S. enterica serotype Enteritidis subtypes identified in the north of Chile. The results of this work demonstrate that genetic diversity, replacement, and expansion of specific S. enterica serotype Enteritidis subtypes were associated with epidemic changes.     

Clinical importance of extended-spectrum beta-lactamase (PER-1-type)-producing Acinetobacter spp. and Pseudomonas aeruginosa strains.

Recently, an extended-spectrum beta-lactamase (PER-1) was found to be disseminated among Acinetobacter spp. and Pseudomonasaeruginosa isolates in Turkey. A population-based cohort study was conducted to elucidate predictive mortality factors in patients with nosocomial infections caused by Acinetobacter spp. and P. aeruginosa, with particular reference to PER-1-type extended-spectrum beta-lactamase (ESBL) production. The study group comprised 16 and 21 non-survivors and 82 and 126 survivors in cohorts infected with Acinetobacter and P. aeruginosa, respectively. In the Acinetobacter-infected cohort, nosocomial pneumonia, hypotension and infection with a PER-positive isolate were independent predictors of mortality. In the P. aeruginosa-infected cohort, impaired consciousness, a PER-positive isolate, male sex and (with a negative relative risk) urinary tract infection were independent predictors of death. This study demonstrated the relationship of PER-1-type ESBL-producing Acinetobacter spp. and P. aeruginosa with poor clinical outcome.     

Localization by insertion mutagenesis of a virulence-associated region on the Salmonella typhimurium 96 kilobase pair plasmid

The virulence-associated plasmid pEX102 of Salmonella typhimurium line TML R66 was tagged with the transposon Tn5 and the virulence of the mutants obtained was assayed in the mouse salmonellosis model. Out of 36 independent insertion mutants tested two isolates had clearly reduced virulence in (CBA x C57B1/6)F1 mice. The corresponding Tn5 elements were positioned on the restriction endonuclease map of pEX102 and found to be some 4 kilobases apart (kb) on the 96 kb virulence plasmid.     

Demonstration of persistence of Salmonella typhimurium in an AIDS patient by molecular methods.

We document microbiological persistence of the same Salmonella typhimurium strain in an AIDS patient during 7 months of clinical observation despite prolonged quinolone therapy. Persistence was demonstrated by phage types that closely resembled one another, similar antibiotic resistance patterns, conserved restriction fragment length polymorphism of chromosomal DNA digested with different DNA restriction enzymes, identical ribotypes, and IS200 types in four characterized sequential isolates of S. typhimurium.     

Comparison of epidemiological marker methods for identification of Salmonella typhimurium isolates from an outbreak caused by contaminated chocolate.

Plasmid profile analysis, restriction endonuclease analysis, and multilocus enzyme electrophoresis were used in conjunction with serotyping, bacteriophage typing, and biochemical fingerprinting to trace epidemiologically related isolates of Salmonella typhimurium from an outbreak caused by contaminated chocolate products in Norway and Finland. To evaluate the efficiency of the epidemiological marker methods, isolates from the outbreak were compared with five groups of control isolates not known to be associated with the outbreak. Both plasmid profile analysis and phage typing provided further discrimination over that produced by serotyping and biochemical fingerprinting. Plasmid profile analysis and phage typing were equally reliable in differentiating the outbreak isolates from the epidemiologically unrelated controls and were significantly more effective than multilocus enzyme electrophoresis and restriction enzyme analysis of total DNA. The greatest differentiation was achieved when plasmid profile analysis and phage typing were combined to complement serotyping and biochemical fingerprinting. However, none of the methods employed, including restriction enzyme analysis of plasmid DNA, were able to distinguish the outbreak isolates from five isolates recovered in Norway and Finland over a period of years from dead passerine birds and a calf.     

Outbreak of Salmonella typhimurium infection traced to contaminated chocolate and caused by a strain lacking the 60-megadalton virulence plasmid.

We describe an outbreak of Salmonella typhimurium infection, caused by contaminated chocolate produced by one Norwegian company, which occurred in Norway and Finland in 1987. A total of 349 bacteriologically verified cases were recorded in Norway, and 12 cases were recorded in Finland. There was a predominance of young children among the patients (median age, 6 years), many of whom developed acute hemorrhagic diarrhea. The outbreak strain exhibited a rare phage lysis pattern and a characteristic plasmid profile lacking the 60-MDa virulence-associated plasmid. DNA hybridization failed to demonstrate any DNA sequence homology between the outbreak strain and the virulence plasmid. The outbreak strain was nonlethal for orally infected mice. The finding of only less than or equal to 10 S. typhimurium cells per 100 g of chocolate in about 90% of the positive samples obtained from retail outlets suggested that an inoculum of fewer than 10 organisms may have been sufficient to cause symptomatic disease.     

Involvement of a Salmonella genomic island 1 gene in the rumen protozoan-mediated enhancement of invasion for multiple-antibiotic-resistant Salmonella enterica serovar Typhimurium

Multiple-antibiotic-resistant Salmonella enterica serotype Typhimurium is a food-borne pathogen that may be more virulent than related strains lacking the multiresistance phenotype. Salmonella enterica serotype Typhimurium phage type DT104 is the most prevalent of these multiresistant/hypervirulent strains. Multiresistance in DT104 is conferred by an integron structure, designated Salmonella genomic island 1 (SGI1), while we recently demonstrated DT104 hyperinvasion mediated by rumen protozoa (RPz) that are normal flora of cattle. Hyperinvasion was also observed in other Salmonella strains, i.e., other S. enterica serovar Typhimurium phage types and other S. enterica serovars, like S. enterica serovar Infantis, possessing SGI1, while DT104 strains lacking SGI1 were not hyperinvasive. Herein we attempted to identify SGI1 genes involved in the RPz-mediated hyperinvasion of Salmonella strains bearing SGI1. Transposon mutagenesis, coupled with a novel reporter system, revealed the involvement of an SGI1 gene previously designated SO13. Disruption of SO13 expression led to an abrogation of hyperinvasion as assessed by tissue culture invasion assays and by bovine challenge experiments. However, hyperinvasion was not observed in non-SGI1-bearing strains of Salmonella engineered to express SO13. That is, SO13 and another SGI1 gene(s) may coordinately upregulate invasion in DT104 exposed to RPz.     

Tn5 mutagenesis of the Salmonella typhimurium 100 kb plasmid: definition of new virulence regions.

In this study, the 100 kb plasmid of Salmonella typhimurium, which is known to contribute to the pathogenicity of the organism, was tagged with the transposon Tn5 to define regions of the plasmid contributing to overall virulence. Eleven randomly selected vir::Tn5 plasmids carried by the plasmid-free S. typhimurium strain WS1321 were physically mapped and then examined in mice for subcutaneous LD50 value, ability to induce splenomegaly, and ability to grow to high numbers in the spleens of infected mice. Nine strains were found to be virulence-attenuated and showed varied levels of growth in the spleens of subcutaneously infected BALB/c mice. Eight of these nine strains carried Tn5 insertions which lie outside the previously defined virulence region. These studies corroborate the findings of other investigators as well as defining novel regions of the 100 kb virulence plasmid involved in the pathogenicity of this organism.     

Molecular epidemiology and antimicrobial susceptibility of Salmonella enterica serotype Stanley isolates in Taiwan.

BACKGROUND AND PURPOSE: Salmonella enterica serotype Stanley became the third most common non-typhoidal Salmonella serotype among human isolates in 2004. The present study was conducted to gain further understanding of the epidemiology and antimicrobial suseptibility of S. Stanley. METHODS: A total of 20 culture-confirmed cases were retrieved from the Center for Disease Control collection and analyzed. Clinical features and demographic data of the cases were analyzed. Laboratory investigation of the isolates included antimicrobial susceptibility testing and molecular typing by pulsed-field gel electrophoresis. Ceftriaxone-non-susceptible isolates were further examined by polymerase chain reaction, sequencing, and Southern blot hybridization. RESULTS: The cases studied were distributed widely across Taiwan, suggesting that the infection was an island-wide problem. S. Stanley predominantly caused infections in patients under the age of 5 years (75%). The most common type of illness was uncomplicated enterocolitis. Molecular typing showed 1 predominant genotype with 5 subtypes among these isolates. Antimicrobial resistance to ampicillin (75%), chloramphenicol (95%), and trimethoprim-sulfamethoxazole (95%) was common. Two isolates expressed non-susceptibility to ceftriaxone, and a bla(CMY-2) gene was identified on an 80-kb plasmid in both isolates. CONCLUSION: The increase in S. Stanley infections may be associated with the spread of an epidemic clone, although this requires further epidemiological surveillance. In view of the high rate of antimicrobial resistance, especially the emergence of resistance to third-generation cephalosporins, continued surveillance of the infections caused by this bacterium should be undertaken.     

肠炎沙门菌引起的一起食物中毒事件的分子流行病学研究

目的对2012年深圳市龙岗区某酒楼发生的一起食物中毒事件的病原体进行分离鉴定和同源性分析。方法对2012年深圳市龙岗区某酒楼发生的食物中毒事件进行现场调查并采集患者肛拭子15份、厨工肛拭子3份和手拭子1份、可疑食品1份以及物表涂抹拭子2份。通过分离鉴定、生化试验和血清学分型确定菌型,再使用脉冲场电泳（PFGE）分型方法对同期患者中相同菌型的菌株进行同源性分析。食物中毒菌株使用K—B进行药敏分析。结果流行病学调查及实验室病原学检测证实本次事件是一起由肠炎沙门菌引起的食物中毒。共分离到16株肠炎沙门菌,其中患者、厨工及可疑食品分离出的肠炎沙门菌同源性达到95％以上,与同一时期散发临床患者分离的肠炎沙门菌同源性为89．4％,可判断为来自同一克隆株。结论此次事件是一起由肠炎沙门菌引起的食物中毒,分子流行病学的证据显示其可能的传播途径为厨工带菌者污染食品烧鸡,被患者进食后引起感染。     

Characterization of genes encoding type 1 fimbriae of Klebsiella pneumoniae, Salmonella typhimurium, and Serratia marcescens.

With a minicell system, the organization of genes encoding type 1 fimbriae of Salmonella typhimurium, Klebsiella pneumoniae, and Serratia marcescens was determined. In all cases multiple gene products were necessary for the phenotypic expression of fimbriae; thus fimbrial expression in these strains is similar to that in Escherichia coli. The type 1 fimbrial subunit gene was detected by the ability of its product to react with specific antiserum. At least six genes were found to be involved in the expression of type 1 fimbriae by S. typhimurium, and at least four genes constituted the fimbrial gene cluster of K. pneumoniae. In the case of S. marcescens, a minimum of three detectable polypeptides was required for the production of fimbriae. Also, a gene probe consisting in part of nucleotide sequences from the E. coli fimbrial subunit gene hybridized to a discrete DNA fragment derived from the plasmid encoding K. pneumoniae fimbriae. Such a fragment was assumed to contain a gene encoding the structural component of the type 1 fimbriae. Each of the three cloned systems encoded a number of polypeptides which varied in size; thus, the organization and molecular weight of fimbrial accessory proteins of each genus were not identical.     

Molecular epidemiology of plasmid spread among extended broad-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital.

Over a 12-month period, 43 children in eight different wards of our hospital (Hôpital Robert Debré) were infected or colonized with Klebsiella pneumoniae strains producing extended broad-spectrum beta-lactamases. The epidemiology of the outbreak was studied by a molecular approach including the determination of the beta-lactamase physicochemical parameters and plasmid profiles, as well as analysis of the restriction fragment length polymorphisms of the rDNA regions (ribotyping). The last approach produced 12 and 5 different patterns with EcoRI and HindIII, respectively, thus identifying 15 different ribotypes among the 43 clinical K. pneumoniae strains. However, 60% of the strains in six wards belonged to only two ribotypes, whereas nine ribotypes were observed only once. Twelve isolates from different wards that were representative of the eight most common ribotypes showed four different beta-lactamase isoelectric focusing patterns and seven different plasmid profiles by direct analysis or after EcoRI digestion. Thus, at least two genetically unrelated strains in the same ward were found to have the same plasmid content. Our results show the complexity of the outbreak, which was associated with patient-to-patient cross-contamination with several epidemic strains with different plasmid contents, interspersed sporadic cases with nonepidemic strains, and the possible spread of a plasmid. The combination of plasmid profile analysis and ribotyping therefore seems to be powerful at deciphering the details of such outbreaks.     

Construction of a recombinant oral vaccine against Salmonella typhi and Salmonella typhimurium.

The viaB gene coding for the Vi antigen of Salmonella typhi Ty2 was subcloned into expression vector pYA248. The recombinant plasmid was termed SMM202 and transformed into Salmonella typhimurium chi 4072, an attenuated delta cya delta crp mutant. Recombinant S. typimurium Vi4072 had the ability to produce Vi capsular polysaccharide and also to invade and colonize the small intestine, mesenteric lymph nodes, and spleen of BALB/c mice. Mice orally immunized with Vi4072 developed serum and secretory antibody responses to the Vi antigen, as measured by a passive hemagglutination assay. Mice developed a delayed-type hypersensitivity following a footpad injection with Vi antigen after being sensitized orally with a suitable dose of Vi4072. Immunization of mice with Vi4072 afforded complete protection against fatal infection with virulent S. typhi Ty2. All data indicate that this route of antigen delivery is effective for stimulating antibody-mediated immunity and for inducing a cell-mediated immune response in BALB/c mice. Thus, S. typhimurium Vi4072 may serve as a vaccine for protection against typhoid fever and salmonellosis caused by S. typhimurium.     

Cloning and transposon insertion mutagenesis of virulence genes of the 100-kilobase plasmid of Salmonella typhimurium.

We have cloned regions of the 100-kilobase (kb) plasmid, pStSR100, of Salmonella typhimurium SR-11 that confer virulence to plasmid-cured S. typhimurium. Cells carrying recombinant plasmids that conferred virulence were selected by inoculating mice orally with recombinant libraries in virulence plasmid-cured S. typhimurium and harvesting isolates that infected spleens. Three plasmids, pYA401, pYA402, and pYA403, constructed with the cosmid vector pCVD305 conferred wild-type levels of virulence to plasmid-cured S. typhimurium and had a common 14-kb DNA insert sequence. Another recombinant plasmid, pYA422, constructed with the vector pACYC184, conferred to plasmid-cured S. typhimurium a wild-type 50% lethal dose (LD50) level, but mice died more slowly than when infected with wild-type S. typhimurium. Furthermore, pYA422 conferred the ability to cause a higher, but not a wild-type, level of splenic infection on plasmid-cured S. typhimurium. pYA422 had a 3.2-kb insert sequence which mapped to the center of the 14-kb common sequence of the cosmid clones. Transposon Tn5 insertion mutations in pYA403 inhibited virulence to various degrees, and when transduced into the native virulence plasmid of S. typhimurium, these Tn5 insertions decreased virulence to degrees similar to those observed when the Tn5 insertions were present in pYA403. vir-22::Tn5 in pStSR100 greatly lowered infection of spleens relative to unmutagenized virulence plasmid, while vir-26::Tn5 and vir-27::Tn5 lowered splenic infection to lesser degrees. At least three proteins were encoded by pYA403 containing 23 kb of insert sequence and subclone pYA420, containing the 14-kb common insert sequence present in all of the cosmid clones. One of these proteins, with an apparent molecular weight of 28,000, was also encoded by pYA422. The Tn5 insertion that most attenuated virulence, vir-22::Tn5, inhibited synthesis of the 28,000-molecular-weight protein. The vir-22::Tn5 insertion was complemented by recombinant plasmids encoding only the 28,000-molecular-weight protein, suggesting a role of this protein in virulence. However, recombinant plasmids, exemplified by pYA422, that encoded only the 28,000-molecular-weight protein did not confer full virulence.